Quantitative protein analysis by immunodiffusion

ABSTRACT

Methods and apparatus are described by which immunochemical procedures such as immunodiffusion and immunoelectrophoresis can be used to provide quantitative measurement of the concentration of individual proteins in fluids such as serum, spinal fluid, tissue extracts and the like. That is accomplished typically by photooptically scanning the zones of precipitation which are produced, in parallel with those of standard preparations containing known concentrations of the proteins to be determined; deriving from the arrays of measurements of the precipitation zones selected parameters; and comparing the parameter values of the experimental and reference preparations. The zone measurements are preferably made and recorded electronically, with suitable digital manipulation of the resulting video values at a plurality of selected positions for each zone.

RELATED APPLICATIONS

This application is a division of application Ser. No. 29,772, filedApr. 13, 1979, which is a continuation-in-part of the copending patentapplication Ser. No. 892,953, filed Apr. 3, 1978, now U.S. Pat. No.4,162,208, which is a division of application Ser. No. 546,351, filedFeb. 3, 1975, now U.S. Pat. No. 4,097,149.

BACKGROUND OF THE INVENTION

This invention relates generally to the quantitative measurement ofproteins in mixtures, especially when only small amounts of material areavailable.

In view of the current rapid expansion of knowledge concerning the roleof proteins in health and disease, there is an increasing need for ageneral, rapid and relatively economical quantitative method for proteinanalysis of such fluids as serum, spinal fluid, tissue extracts and thelike.

The proteins occurring in such fluids are frequently identified byimmunochemical procedures which depend upon precipitation of eachprotein by an antibody specific to the particular protein. Theproduction of such specific antibodies is stimulated when foreignproteins (antigens) are introduced into a living body. Antisera can beprepared, containing known mixtures of such antibodies. By reacting aprotein sample in vitro with such an antiserum and observing theresulting precipitation, or lack of precipitation, useful qualitativeinformation may be obtained as to the types of proteins in the sample.

SUMMARY OF THE INVENTION

A primary object of the present invention is to obtain quantitativevalues for the concentration of one or more proteins in an antigensolution, utilizing immunodiffusion procedures which have previouslybeen regarded as only qualitative techniques. That has been accomplishedin large part by selecting suitable parameters for measurement andcomputation, and by providing adequate reference standards to which theresults can be compared.

In preferred form of the invention, the proteins in the initial antigenmixture are first partially fractionated by causing them to migrate inone dimension at rates that differ characteristically among the variousproteins. Such selective migration may, for example, utilize simplediffusion, electrophoresis, or more complex techniques such aschromatography. In the illustrative case of electrophoresis, differencesof electrophoretic mobility between different proteins cause theproteins to become distributed in the direction of the electrical fieldin accordance with their mobility. Following such initial fractionation,the resulting essentially linear distribution of proteins is broughtinto contact with the antiserum by relative movement in anotherdimension, typically by mutual diffusion in a suitable agar or agarosesupport medium. The precipitation zones of the respective proteins arethen typically entirely separate and can be clearly distinguished. Thatoverall process, known as immunoelectrophoresis, is well recognized as aqualitative method of great power and flexibility for detecting thepresence or absence of certain antigens or the antibodies against them.

Useful separation of the precipitation zones of a plurality of distinctproteins is also attainable without an initial step of fractionation, ifthe antigen and antibody are allowed to diffuse toward each other in amanner to form elongated precipitation zones of limited length extendingtransversely of the primary direction of diffusion. The mobilities fordiffusion of different proteins, and/or of their antibodies, areordinarily sufficiently different that such precipitation zones areclearly distinguishable. Any overlapping that may occur is usuallylimited to portions of the zones, the zone end points being usuallyclearly separated. The quantitative procedures of the present inventionare usefully applicable to immunodiffusion of the described type.

Similarly, if antigen and antibody are placed into wells forimmunodiffusion, and an electric field is applied to accelerate themovement of the antigen and antibody toward each other, as in theprocedure known as electroimmunodiffusion, the resulting precipitatezones retain the same basic forms as in absence of an electric field.The same is true if the immunodiffusion step of immunoelectrophoresis isaided by a suitably directed electric field. Accordingly, the term"immunodiffusion" in the present specification and claims refers todiffusion with or without an accelerating electric field.

In accordance with one aspect of the invention, a large number ofmeasurements are carried out in a systematic manner as the precipitationpattern develops, and the resulting direct data are then employed forderiving values of carefully selected and relatively specific parametersof individual selected precipitation zones. Those parameter values arecompared with values obtained under comparable conditions from a seriesof standard or reference runs which have been suitably selected andtreated to facilitate reliable interpolation. That procedure has beenfound to yield satisfactorily consistent and reproducible quantitativevalues for the actual concentrations of the selected proteins in thesample.

The described collection of initial experimental data and thecomputations made with them can be carried out manually, if desired.They are also well adapted for semi-automatic data collection by opticaland electronic scanning devices. Also, the necessary data processing canbe made fully automatic by use of a general purpose computer of moderatecapacity. Such automated operation permits collection and processing ofample data for calculating results from several independent sets of dataor by use of more than one parameter or group of parameters, therebyproviding a basis for estimating reliability, as by computing probableerrors.

A further aspect of the invention employs comparisons of concentrationvalues obtained from different parameters, or evaluation of parametervalues themselves, as a means of detecting presence of certain proteinabnormalities.

The invention further may utilize the same optical sensing system forscanning the precipitation zones and for initially locating andidentifying the reactant wells for automatically depositing selectedreactants in them.

BRIEF DESCRIPTION OF THE DRAWING

In the following description of certain illustrative manners of carryingthe invention into practice, reference well be made to the accompanyingdrawings in which:

FIG. 1 is a schematic plan of an immunoelectrophoresis plate, FIG. 1Aillustrating a typical distribution of proteins followingelectrophoresis, and FIGS. 1B, 1C and 1D illustrating successive stagesof the subsequent diffusion and immunoprecipitation reaction;

FIG. 2 is a schematic axial section representing typical apparatus formeasuring a slide;

FIG. 3 is a schematic drawing illustrating properties of a precipitationzone relating to the invention;

FIG. 4 is a graph representing typical dependence of zone end pointsupon time;

FIG. 5 is a graph representing typical dependence of zone length upontime;

FIG. 6 is a graph representing typical dependence of time of initialzone appearance upon protein concentration;

FIGS. 7 and 8 are graphs representing typical dependence of zone lengthupon time and upon protein concentration;

FIG. 9 is a graph showing actual intensity scans across a precipitationzone at two incubation times;

FIG. 10 is a graph representing intensity scans across precipitationzones formed by respective protein concentrations;

FIG. 11 is a graph representing illustrative dependence of the parameterL_(z) upon time;

FIG. 12 is a graph representing illustrative dependence of the parameterI_(z) and of the parameter dI_(z) /dt upon protein concentration;

FIG. 13 is a schematic graph illustrating derivation of proteinconcentration values by known additions of the protein to the unknownsample; and

FIG. 14 is a schematic block diagram representing electronic scanningapparatus for carrying out the invention.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Many aspects of the invention are well illustrated by its embodiment inthe preferred process of immunoelectrophoresis, and the followingdescription will emphasize that form of the invention, but withoutimplying any limitation of scope.

Immunoelectrophoresis is well known as a qualitative procedure, and manyforms of apparatus have been described for carrying it out, differing indetail rather than in principle. The electrophoresis and subsequentdiffusion and immunoreaction are typically performed in a single layerof gel from a fraction of a millimeter to several millimeters thickcarried on an optically transparent sheet. A currently preferredsupporting medium is agarose saturated with barbital buffer ofapproximately pH 8.6 and ionic strength 0.1. The active materials aretypically inserted into wells, which may be cut from the gel coating orformed when the gel is molded on the carrier.

FIG. 1 shows schematically a plate 20 with a typical arrangement ofwells, comprising the two circular antigen wells 21 and 22, spacedequally on opposite sides of the elongated antibody well or trough 24with axis 25, which extendss parallel to the direction ofelectrophoresis. With that geometric arrangement of wells two distinctantigen solutions, or two specimens of the same solution, can be runsimultaneously against the same antibody solution on each plate. Ifpreferred, the capacity of each plate can be multiplied, as by providingadditional antibody wells outward of the two illustrated antigen wells,with additional antibody wells outward of them. Similarly, additionalantigen wells may be added, spaced far enough from wells 21 and 22 inthe direction of electrophoresis to prevent overlapping of the patterns.

The shaded areas 26 and 28 in FIG. 1A represent typical approximatedistributions of four varieties of protein a, b, c and d from duplicatesamples in the respective wells 21 and 22 following a period ofelectrophoresis in the direction of the arrow 23. Although all proteinsusually migrate in the same direction through the liquid medium, thesolvent itself tends to carry a net charge and to have a resultant flowor electroosmosis relative to the gel. Hence the proteins may have a netmovement in either direction relative to the well.

Following termination of electrophoresis and addition of antibodysolution to well 24, precipitation zones are produced by mutualdiffusion of the proteins a, b, c and d of FIG. 1A and the correspondingantibodies. FIGS. 1B, 1C and 1D show typical zones at respective stagesof development.

The precipitin arcs of unrelated antigens, reacting with the antibodiesagainst them, form independently, and may cross when they aresufficiently close together, as shown in FIG. 1D; whereas the arcs ofantigens that are immunochemically related typically join in acontinuous reaction. The zone of precipitation d' in FIGS. 1C and 1Dindicates that the area d of FIG. 1A actually contained two distinctproteins, illustrating the fact that even antigens which have identicalelectrophoretic mobility may form distinct zones of precipitation. Thezones d and d' may alternatively be viewed as representing two distinctproteins that were initially placed in well 21 and were subjected toimmunodiffusion without initial electrophoresis. The clear separation ofthe respective zone ends would then be due to different rates ofdiffusion of the proteins or of their antibodies. In either case, eachsuch zone can be analyzed independently, using any or all of the methodsof analysis to be described.

In accordance with the present invention, the zones of precipitationresulting from immunoelectrophoresis are subjected to directquantitative measurements. Such measurements may determine only thephysical position on the plate of certain selected features of eachprecipitation zone of interest, or may include quantitative photoopticalmeasurements of the light intensity. For both types of measurement, thetime is noted and may be used as an integral part of the observationaldata. If the incubation process is allowed to reach equilibrium,producing a nearly static zone configuration, the exact time ofmeasurements becomes immaterial.

Position measurements on slide 20 can be made, for example, with the aidof a low power microscope. As represented schematically in FIG. 2, plate20 is placed over an adjustable opening 32 in the top of a light-tightbox 30 with the lamps 36 and the dark backing 34 of light absorbingmaterial such as black velvet. The microscope 40 has the objective lens41, the eye piece 42 and a set of cross hairs or other reference reticleat 43 in the focal plane. The microscope is mounted above light box 30on a double slide mechanism 45 with screw drive 46, and with accuratescales, not explicitly shown, for reading the microscope position in twocoordinates. For clarity, only one coordinate of the movement isdirectly indicated in the drawing. It is usually convenient to selectcoordinates having the x-axis, say, parallel to the direction ofelectrophoresis and to the length of the antibody well, and having theorigin of coordinates at or near the well axis 25, as indicated in FIG.1A.

For measurements of the light intensity the microscope typicallyincludes an oblique beam-splitting mirror 48 which sends part of thelight to the eye piece while another part forms a real image in theplane of a diaphragm 52. Diaphragm 52 then transmits to thephotosensitive transducer 50 only radiation from the elemental area ofplate 20 that coincides optically with the cross hair image. Transducer50 is electrically connected to the amplifying circuit 54 and the meter56. The latter may be observed visually and the value recorded manually,or the meter may embody means such as analogue-to-digital circuitry andprintout mechanism for automatically recording the light intensity inresponse to a command signal. Many changes can be made in theillustrative apparatus of FIG. 2, including, for example, replacing thedark field illumination by direct lighting or by top lighting such thatlight reflected from the zone is observed or measured. Illustrativeapparatus for making completely automated measurements is describedbelow.

FIG. 3 illustrates schematically certain preferred precipitation zonefeatures which are selected by the present invention for positionmeasurement as the zone develops. The horizontal line 61 at y=y_(Ab)represents the adjacent edge of the antibody well in theimmunoelectrophoresis slide. The points E and F at the coordinates(x_(e), y_(e)) and (x_(f), y_(f)) respresent the left and right endpoints of the elongated zone 60.

In addition to the zone end points E and F, we have found it useful tomeasure coordinates of a number of intermediate points of each zone. Thesingle point G at the coordinates (x_(g), y_(g)) in FIG. 3 isillustrative of such points.

Since the zone has some width in the y direction, the y coordinate ofeach intermediate point such as G can be conveniently placed either at63 at the leading edge of the zone closest to the antibody well, at 65at the trailing edge of the zone, or at one or more points such as 64within the zone, typically including the point of maximum intensity.

As the zone develops with increasing time of incubation, the points E, Fand G progressively change both their relative and their absolutepositions. Illustrative values of x_(e) and x_(f) as functions of timeare plotted in FIG. 4 for a typical protein concentration. Such positionvalues may be used directly for determining protein concentration, as bycomparing the values of x_(e) and x_(f) obtained with unknown solutionsto corresponding values obtained with known concentrations of proteinsat measured times. However, more reliable and accurate results areordinarily obtainable by deriving from such initial measurements one ormore functions, which will be referred to for convenience as parameters.

An important parameter of the precipitation zone employed by the presentinvention is the coordinate difference x_(f) -x_(e), which is a measureof the length L of the precipitation zone at the particular time t ofmeasurement. The variation of that parameter with time of incubation isplotted in FIG. 5 for the typical data of FIG. 4.

Position parameters other than the zone length L may be computed frommeasurements of the zone as it develops. For example, the zone curvatureand its variation along the length of the zone are useful parameters, aswell as providing information as to presence of protein abnormalities(see below). A rough measure of curvature for the zone arc as a whole,or for a selected zone segment, can be obtained by relatively simplecomparisons of the x and y coordinates for three mutually spaced pointson the zone axis. To obtain more accurate values of zone curvature, thezone axis is typically fitted approximately by a curve of the typey=f(x), where f(x) may represent any suitable function of x. The radiusof curvature R is then given by the general formula ##EQU1## where y'and y" represent the first and second derivatives of y with respect tox.

A suitable illustrative function for curve fitting represents aparabola, typically with axis parallel to the y axis. Such a parabolamay be expressed in either of the equivalent forms

    y=a.sub.o +a.sub.1 x+a.sub.2 x.sup.2                       (2a)

and

    y=A(x-B).sup.2 +C                                          (2b)

where

    A=a.sub.2 B=-a.sub.1 /2a.sub.2 C=a.sub.o -a.sub.1.sup.2 /4a.sub.2

The values of the constants in (2a) or (2b) can be found directly fromthe coordinates of any three points of the zone axis, or may be fittedby least squares or other known procedure to any desired number of suchpoints. The axis of symmetry of the parabola is at x=B, and the curve atthat axis is a distance C from the x axis. Using the formula (1), theradius of curvature may be expressed as ##EQU2## That radius has itsmaximum value R_(o) at the axis of symmetry, where (3) reduces to

    R.sub.o =2A                                                (4)

Any of the above quantities, which may be derived as indicated from asfew as three points of the zone axis, may be employed as parameters inaccordance with the invention.

Another parameter that is useful for determining protein concentrationin accordance with the invention is the time T_(o) of first appearanceof the zone. That time is difficult to determine by direct observation.One aspect of the invention provides a practical way of obtaining areliable and reproducible value for the time of first appearance.

In FIGS. 4 and 5 the solid lines represent typical plots of directexperimental values. The figures also include extrapolations of thesolid curves toward earlier times. The extrapolations are shown asdashed lines. The point 66 at which the extrapolated curves of FIG. 4meet represents a time at which the zone must have had zero length. Theextrapolated curve of FIG. 5 intersects the time axis at the point 67,giving an equivalent procedure for finding T_(o). Such extrapolationrepresents a reasonable and highly useful definition of the time offirst appearance of the precipitation zone. That method of determiningT_(o) has the advantage that continuous observation of the plate is notrequired.

Turning now to the quantitative determinations of the zone lightintensity, it has been discovered that a single light intensity readingdoes not usually provide a useful measure of protein concentration. Thatis primarily due to the variability of zone shape and speed offormation, and the tendency of the intensity to change as the zoneexpands.

On the other hand, we have found that the variability of such factorscan be largely compensated by taking a series of intensity readings atsuitably selected locations and treating them collectively to evaluatean intensity parameter. A preferred procedure is to take such readingsat uniform intervals along a line extending linearly across the zone ofprecipitation, typically in the y direction at a particular value of x.Such a series preferably includes several intensity values outside ofthe zone at each side. Those offset values are then averaged to providea measure of the background intensity, which is subtracted from each ofthe intensity readings within the zone. The resulting adjusted intensityvalues are effectively summed, yielding essentially a linear integral ofthe intensity along a line crossing the zone at a selected value of x.We have found that such a linear intensity sum I_(x) tends to increasewith incubation time in a regular and reproducible manner, the value atany given time increasing with the concentration of the reacting proteinover a wide range of experimental conditions.

Typical plots of the relative intensity observed during cross-zone scansare shown in FIG. 9. The two curves were plotted by semi-automaticapparatus of the type described below, scanning in the y direction atx_(g), the point of closest approach of the precipitation zones to theantigen well. The peaks at 74 and 77 in FIG. 9 are due to the zonesformed on opposite sides of an antibody well by identical proteinsamples in the two antigen wells of a plate similar to that of FIG. 1.The two small peaks at 75 and 76 are due to the respective edges of theantibody well, providing a convenient reference from which to measuredistances such as D from selected zone points to that well. Theparameter I_(x) defined above corresponds essentially to the area undera peak such as those at 74 or 77 of FIG. 9.

Peaks 74 and 77 of FIG. 9 were made with identical samples of humanserum albumin. They illustrate typical development of the precipitationzones between 2 hours of incubation (solid line curve 70) and 4 hours(dashed line curve 72). Although the zone position remains remarkablystationary during the time between those two sets of measurements, thearea of each peak grows appreciably. The near identity of the peaks at74 and 77 is noteworthy. The two curves are offset vertically by anarbitrary distance for clarity of illustration.

FIG. 10 is a schematic plot illustrating typical scans in the ydirection on plates made with different concentrations of protein, allmeasured at substantially the same time of incubation. The graph bringsout clearly the increasing area of the individual peaks and the shift ofthe entire zone toward the antibody well with increasing proteinconcentration progressing from peaks A to C.

Whereas the parameter I_(x) is highly useful for determining proteinconcentrations, still better results are obtained from a multipleintensity parameter, obtained by summing or averaging such linearintensity sums at several different x values. A typical procedure is tocompute linear sums at x_(g) and at values spaced on each side of x_(g)by a selected interval. Averaging or summing a uniform predeterminednumber of such linear sums reduces experimental error and improves theoverall accuracy of the determination of protein concentration.

In accordance with another, generally preferred procedure, each time theplate is scanned the number of linear sums included in the computationis increased as the length of the precipitation zone increases. Anillustrative procedure of that type is to determine the linear sum ofthe intensity at x_(g) and to continue to compute such sums on each sideof x_(g) until the value of the sum falls below a selected threshold.Addition of all the linear sums provides a parameter I_(z) which isessentially the integral of the intensity of the zone of precipitationat the time of the scan. That approximation can be obtained as preciselyas is desired, within the limits of resolution of the instrumentation,by reducing the x and y increments at which measurements are made. Thevalue of I_(z) varies especially steeply as a function of proteinconcentration over a wide range of experimental conditions. That isbecause, as the concentration is increased, both the x and y dimensionsof the zone increase, and the average intensity of the zone also tendsto increase. That steeper dependence upon protein concentration makesthe total intensity parameter especially effective as a criterion fordetermining the concentration.

After obtaining experimental values for one or more parameters for anantigen sample to be analyzed, those values are compared with suitablesets of standard values for the respective parameters, obtained underclosely similar experimental conditions but with a series of proteinsolutions containing respective known concentrations of the protein ofinterest. To obtain such an array of standard values, standard runs arecarried out with such standard protein solutions, and measurements aremade at corresponding points of the respective plates at successivetimes as their incubation proceeds. The standard runs are preferablycarried out with all conditions as nearly identical as possible to thoseof the experimental runs for which they are to provide reference values.In fact, a distinct set of reference values is preferably obtained forevery group of experimental runs. However, for routine measurements forwhich the reference curve slopes are known from previous experience,even a single standard run may sometimes suffice.

Standard values of the selected parameter are derived from the resultsof those standard measurements, typically for each concentration and atseveral times. Each measured standard value of the parameter is thereforconsidered as a function of both concentration and time. When individuallinear intensity sums I_(x) are to be considered separately, a fullidentification also requires specification of the value of x.

An advantage of using the time of first appearance of the zone as aparameter is that standard values of T_(o) do not involve time as avariable. That is, although the evaluation of T_(o) by the methodsdescribed requires measurements at a series of definite times, onceT_(o) has been derived from those measurements the times of therespective measurements become immaterial. Thus, the values of T_(o) canbe plotted as a function of protein concentration C, yielding a singlestandard curve. Such a curve is represented schematically in FIG. 6,typically based on values obtained for respective concentrations by theextrapolation method described in connection with FIGS. 4 and 5.Concentration values are directly readable from the curve of FIG. 6 foran unknown sample once its T_(o) has been measured.

In the case of parameters such as L, I_(x) and I_(z) preparation ofstandard curves may be less direct. Since values depend upon the timesat which the measurements are made, the series of reference standardsmust be prepared in such form that they cover a range of times. It isnot always feasible to measure data for all concentrations at the samemoment. Each measured value is therefore associated with its time ofmeasurement, and the resulting standard values for the respectiveprotein concentrations are then plotted on separate curves as functionsof time.

FIG. 7 shows a typical family of such curves in which standard values ofthe parameter for three concentrations are plotted against time. It isnoted in passing that the indicated extrapolation of those curves to L=0can provide standard values of T_(o) for plotting FIG. 6, or can provideexperimental values of T_(o) for comparison with FIG. 6. Vertical linesare drawn on FIG. 7 at a series of arbitrary times, shown as t₁, t₂ andt₃. Their intersections with the curves then provide a set of L valuesfor different concentrations, all corresponding to the same time. Eachsuch set of L values is replotted as a separate curve as a function ofconcentration. The result is an array of curves, each showing L as afunction of protein concentration for a particular time. Such an arrayis shown schematically in FIG. 8, and is found more convenient forcomparison with an experimental parameter value than the plot againsttime of FIG. 7.

Standard values for comparison with experimental values of otherparameters are typically obtained and treated in a manner analogous tothat described for the parameter L.

We have discovered, however, that the total intensity parameter I_(z),when measured for given protein concentration at successive times ofzone development, increases linearly with the time. That linear relationis illustrated in FIG. 11, which is a plot of I_(z) against time forfour solutions containing the indicated known concentrations of theprotein immunoglobulin. The indicated values of I_(z) were derived by asuitably programmed general purpose computer from intensity valuesmeasured automatically at a two-dimensional array of zone poositions inthe general manner to be described. The points shown in FIG. 11 wereoriginally plotted automatically, and the straight-line curves werefitted to each set of points, by the computer. The figure has beenredrawn manually and is reproduced at greatly reduced scale.

The linear relation shown in FIG. 11 aids the preparation of standard orreference plots from which to read the protein concentrationcorresponding to an experimental value of the parameter I_(z). One suchcurve, derived from the data of FIG. 11 and showing I_(z) as a functionof concentration for the time 2.4 hours, is shown in FIG. 12 at 70.

The linear dependence of I_(z) upon time also means that the timederivative dI_(z) /dt, or rate of change of I_(z) with time, isconstant. It thus provides a parameter having the practical advantagethat it does not depend upon time. Curve 72 in FIG. 12 illustratestypical behavior of that parameter as a function of proteinconcentration, each point representing the slope of one of the curves ofFIG. 11. As already explained in connection with the parameter T_(o) andFIG. 6, a single curve such as 72 of FIG. 12 can serve effectively asreference curve for the parameter dI_(z) /dt.

A further aspect of the invention provides improved accuracy, especiallywhen the sample under study contains the protein or proteins of interestat relatively low concentrations. The standard solutions are thenpreferably supplemented by adding known amounts of such proteinsdirectly to aliquot portions of the sample itself, and running theresulting solutions in parallel with the original solution, and withstandards containing definitely known concentrations of the protein.Values are obtained for the desired parameter for the respectivesolutions all at a uniform time, using the above described technique forinterpolating with respect to time if necessary. The parameter values Pobtained for the regular standard solutions are plotted against proteinconcentration in the usual way, as indicated schematically by curve 80in FIG. 13. Also plotted are the values of P for the original antigensample, and for the portions of that sample to which protein was addedin known amount, indicated typically as the respective points h, i, jand k. Those points are plotted relative to the horizontal concentrationaxis as if the solutions contained only the protein that was added. Thusthe value h for the original sample is plotted at C= 0. Curve 81 isdrawn through those points. With that illustrative arrangement of thedata, the protein concentration in the original sample can be evaluatedin several ways, which should give essentially the same value. Thus, anydesired number of those procedures may be used, and the resulting valuesaveraged.

First, horizontal projection from point h to intersect curve 80 at h'yields the concentration value indicated at C_(h), which corresponds tothe general comparison procedure previously described. Further, similarprojection of each value i, j and k to intersect curve 80 provides ameasure of the concentration in terms of the lengths of the lines from ito i', j to j' and k to k', all of which lengths are theoreticallyequal. If the value of P at he is somewhat uncertain, for examplebecause the observed zone is faint, an average of all four intervalsgives a more reliable value.

Another procedure has the advantage that it can not only improve theaccuracy of an uncertain determination of h, but can also provide ananswer even if the original sample contains less than the thresholdvalue of protein needed to produce a measurable precipitation zone. Inthe latter case, the upper portion of curve 81 is drawn through theavailable points i, j and k, and is extrapolated to the P axis, thusproviding a determination of P at h. In thus projecting curve 81,standard curve 80 is a helpful guide. For example, curve 80 is shiftedbodily to the left in FIG. 13 till it represents as well as possible thepoints i, j and k. The intersection of the shifted curve 80 with the Paxis then gives a good measure of point h, from which the concentrationC_(h) is found. Also, further extrapolation of curve 81 to the negativeC axis at -C_(h) gives a direct reading for the concentration, whichshould agree with the value C_(h).

The described reliance upon the supplementary standard curve 81 has thegreat advantage of using the identical medium for the experimental andreference solutions. Especially when that medium is the human serum of aparticular patient, that advantage more than overcomes any possibilityof slight error in extrapolating the standard curve. By increasing thenumber of different amounts of added protein, the three shownillustratively in FIG. 13 being merely illustrative, and by usingseveral runs for each amount, the reliability of the extrapolation canbe increased almost without limit, and a good indication can be obtainedof the experimental error that it may involve.

The invention further provides methods for the automated detection ofabnormalities of certain proteins in an antigen sample. Normal andabnormal gamma globulin, for example, typically have slightly differentranges of electrophoretic mobility, but react with the same antibody,leading to precipitation zones with irregular and typicallyunsymmetrical distribution of precipitate along the length of the zone.Such irregularity can be detected by comparing experimental values ofthe intensity parameter I_(x), for example, for different values of x.Observation of unsymmetrical or otherwise irregular variation of suchvalues indicates that an abnormality has been encountered; and thatindication is greatly strengthened if several independent determinationsare carried out at each x value and if the computed experimental errorsshow the variation to be statistically significant.

That method may be considered as a special case of the general procedureof comparing the experimentally obtained values of different parameterswhich tend to respond differently to the abnormality of interest. Forsome parameters with such behavior it may be more effective to comparethe values of protein concentration that correspond to the respectiveobserved parameter values, rather than directly comparing the parametervalues themselves. For example, the described irregular intensitydistribution along the zone in presence of abnormal gamma globulin tendsto cause the zone to appear sooner than normal, leading to higher valuesof computed concentration; whereas the total intensity parameter I_(z)tends to yield approximately equal concentration values for the normaland abnormal proteins. Thus significantly poor agreement betweenconcentration values obtained with T_(o) and with I_(z) indicatespresence of abnormal protein.

A further parameter which responds sensitively to presence of abnormalprotein is the curvature of the longitudinal axis of the zone. Inpresence of abnormality the curvature tends to vary irregularly andunsymmetrically along the length of the zone, while the overallcurvature tends to be less than normal.

Further advantages are nearly always obtainable by employing multipleparameters which comprise specific functions of two or more parameters,such as the described position or intensity parameters. For example, thesum of the zone length L and an intensity parameter, each appropriatelyweighted according to its experimental error of measurement, provides anew parameter which tends to give more reliable results than either oneof its components when used alone.

Another advantageous combination comprises a differential function oftwo parameters, one of which increases with increasing proteinconcentration, while the other decreases. Thus, a quotient or adifference of such parameters provides a multiple parameter with asteeper dependence upon concentration than either of its components. Thetime T_(o) of first appearance of the zone is an example of a parameterhaving inverse relation to protein concentration, and is especiallyuseful for constructing such quotients. The distance from the antibodywell to the zone at any desired value of the x coordinate, for examplethe distance of closest approach at x_(g), also depends inversely uponthe concentration and may be employed as a parameter in such quotients.It is generally preferred to divide the parameter with direct dependenceupon concentration by that with inverse dependence, so that theresulting multiple parameter has a direct rather than inverse netdependence.

Suitable reference values for comparison to experimentally determinedmultiple parameters of unknown samples are typically derived fromreference values obtained as already described for the respectivecomponent parameters.

Whereas the position and intensity measurements and the parameterderivations that have been described can be carried out by direct visualand manual operations, as has been indicated, an advantage of theinvention is that those operations are particularly well adapted forpartial or substantially complete automation.

An especially convenient and effective procedure for making themeasurements required by the invention is to scan the slide by opticalmeans, such as a television camera, a charge coupled scanning device orequivalent means, for producing a video signal representing the apparentbrightness of the scanned image at a two-dimensional array of elementalareas. That signal for each element is typically converted to digitalform and electronically stored in association with signals representingthe x and y corrdinates of the corresponding position on the plate andthe time of observation. The entire array of such position signals, orthe signal for a designated position, can then be recovered from memoryas needed for further processing. Also, any desired portion of the slideimage can be displayed either during the scanning operation or at alater time by means of a cathode ray tube or equivalent display device.Systems for scanning, storing and reproducing an image, and forextracting a video signal in digital form for a selected image point arewell known in the electronic art, and are available commercially informs well adapted for the present purpose.

FIG. 14 represents schematically such apparatus in illustrative form,comprising the television camera 90 with lens 91 for imaging the plate20 on the photosensitive surface of the camera, the monitor cathode raytube 92, the scan control apparatus 94 and the general purpose computer100. Plate 20 can be imaged at any desired magnification by conventionaladjustment of lens 91. A desired portion of the plate can be centered inthe field by shifting its position on its illuminated support, such aslight box 30 of FIG. 2, or by conventional electronic bias controls inthe camera circuits.

More particularly, the scanning movements in the scanning device and inthe synchronized monitor cathode ray tube, if used, are typically drivenstepwise in both coordinates, with steps so small that the movementappears virtually continuous on the screen. The image is thereby dividedinto elemental areas, which can be identified, for example, byspecifying their x and y coordinates. For manual or semi-automaticoperation, the monitor display typically includes a cursor comprising anelectronically produced bright spot which designates on the screen theelemental area from which the video sample is being extracted once eachcomplete scan. The operator is provided with manually controlledelectronic switching means by which he can move the cursor about on thescreen under visual control. Means may also be provided for directlyplacing the cursor at a desired point, typically in response to x and ysignals in digital form constituting a command address. Such signals maybe derived under manual control, or supplied from a computer in responseto conventional program instructions.

As illustratively shown in FIG. 14, the counter 102 counts pulsesreceived from the clock 103, producing on the multiple lines 104 digitalsignals which represent successive x coordinate values, say. Circuit 106develops on the line 107 an analogue step voltage in response to thosesignals, with a step corresponding to each elemental x value. Thedividing circuit 108 effectively counts the beam sweeps in the xdirection, producing on the multiple lines 109 digital signalsrepresenting the y coordinate for each sweep. Circuit 110 develops onthe line 111 an analogue step voltage in response to that count, with astep corresponding to each elemental y value. The step voltages on lines107 and 111 control the x and y sweep movements in camera 90 and tube92, insuring their synchronization. The video signal from camera 90 issupplied via the line 112 to monitor tube 92, reproducing on the tubescreen the brightness variations of slide 20.

The selection circuit 118 typically comprises x and y counters which areselectively shiftable up or down either by a single counter or in acontinuing series of counts in response to manual movement of the joystick indicated schematically at 119. The resulting digital signals,representing x and y coordinates of a point selected for exploration,are stored in register 117. The comparison circuit 114 continuouslycompares the coordinates of that selected exploration address fromregister 117 with those of the scanning beams from lines 104 and 109.When the scanning spot reaches the stored address, typically once duringeach complete scan, comparison circuit 114 supplies an enabling pulse tothe switching circuit 120. The video signal from camera 90 is therebytransmitted to analogue-to-digital circuit 122. The register 125 thusreceives on the lines 123 a digital representation of the brightness ofthe scanned slide 20 at the selected exploration point. The enablingpulse from circuit 114 is supplied also to the cursor control circuit126, superimposing upon the video signal a beam intensifying pulse whichidentifies that selected point on the monitor screen, as indicatedschematically at 127.

Register 125 also receives the x and y coordinate signals from lines 115and 116 and a digital signal continuously representing the time,developed under clock control by the time circuit 128. All of thosesignals are typically stored in register 125 for delivery to computer100 in response to unload signals which may be supplied either undermanual control via the line 129 from selector 118 or via the line 130under control of the computer.

Computer 100 is preferably provided with means for independentlydesignating an exploration address and thus obtaining input informationfor the spot of plate 20 that corresponds to specific requirements ofany program under which it is operating. Such exploration means maycomprise circuitry basically similar to selector 118, register 117 andcomparison circuit 114, the selector operating in response to electronicsignals which selectively indicate the required direction of movement ofthe exploration spot or the digital address of its required position.Rather than duplicating such apparatus, FIG. 14 indicates computercontrol of selector 118 by electronic signals supplied via the multiplelines 132, supplanting joy stick 119 when the switch 133 is shifted frommanual position to automatic. With such control available the computeris readily programmed to carry out position and intensity measurementsand parameter derivations of the general types that have been described.

For automatic scanning of zones, the plates of a set may be transferredin sequence from the incubation chamber to an accurately definedscanning position with suitable illumination. Especially when theoptical scanning means is both light in weight and compact, as in thecase of a charge coupled device, for example, it is generally preferredto mount that scanning device on a platform that is movable in twodimensions over the stationary array of immunodiffusion orimmunoelectrophoresis plates. That arrangement facilitates use of thescanning system as an aid to fill the antigen and antibody wells. Forthat purpose a digitally controlled dispenser is mounted in fixed butoffset relation to the scanning device. Video signals from the scannerare supplied to the computer, which is suitably programmed to recognizethe shapes of the individual wells, or to identify machine readablesymbols associated with them. As the platform carrying scanner anddispenser is moved under computer control over the plates, it can thenreadily be stopped when the scanner axis is located accurately over aselected antigen well. The platform may then be moved a fixed incrementto center the dispensing tip over the well so that the correct charge isdispensed accurately into the well.

When all wells are filled in this manner with the appropriate antigensolutions, with suitable washing of the dispenser between operations,electrophoresis is initiated. A similar loading operation is typicallycarried out to charge the antibody wells, either after completion of theelectrophoresis, or immediately after filling the antigen wells ifelectrophoresis is omitted. After the desired time for diffusion thesame scanning device is moved successively to all positions where zonesof precipitation are to be scanned throughout the array of plates.

The scanning capability of the scanning device is preferably used alsoto scan the array of plates before the wells are loaded. The computer isprogrammed to compare the observed positions of the antigen and antibodywells, recording any departure from uniformity of dimensions or relativepositions among the plates of the array. Any plate or individual wellfound to depart too far from standard can then be automatically omittedduring the loading operation; and smaller deviations can be compensatedby automatically applying suitable corrections to the parameter valuesthat are ultimately derived by the computer.

Initial zone scanning procedure typically comprises request by thecomputer for video information at successive exploration addresses witha selected x value and with y values shifting progressively by aspecified interval over the region in which zone formation isanticipated. The video intensity values received from register 125 arestored with x, y and t data for each explored point. After each suchy-scan, the x coordinate is shifted by a specified interval and asimilar y-scan is performed and the results recorded, until the entirearea of interest has been covered. Each received intensity value istypically compared with the previously received and stored values. Ifthe observed intensity variations exceed a set threshold characteristicof a background area, indicating presence of a precipitation zone, eachintensity value within the zone is so designated in memory. Also, foreach y-scan, the intensity maximum as the zone is crossed is identifiedand recorded, establishing the zone axis in terms of a series of yvalues at uniformly spaced x values. The end points of each zone areidentified typically as the terminal x values in each direction at whichan intensity maximum was identified. If more precise location of the endpoints is desired, the computer is instructed to perform further scansin a defined region about each end point with reduced x and y intervals.

With the existing precipitation zones so located and recorded,straightforward arithmetic operations are performed on the stored data,yielding the described intensity sum parameter I_(x) for each cross-scanof a zone in the y direction; and addition of those values gives thetotal intensity parameter I_(x) for the zone. Subtraction of the xvalues at the zone ends gives the parameter L. Additional parameters maybe obtained by appropriate computation as desired.

The described measurements are preferably carried out as a unitaryoperation on a complete set of plates that includes one or more unknownsamples and also a set of related standard solutions of the typediscussed above and sufficiently complete to permit evaluation of theunknown samples. After each scan of such a plate set, the computer ispreferably directed to derive concentration values for each of theproteins for which precipitation zones were found. If, as is preferred,multiple standard runs are included, the probable experimental error canbe derived for each calculated concentration value. Computer routinesfor such calculations are well known. Several independent determinationsare preferably made of the concentration of each protein in the sample,typically by reference to different parameters; an average is thencomputed, with each value weighted in the usual way according to itsprobable error.

If the computed probable error for that average is within the specifiedrange for the particular sample under study, no further measurements areneeded. The computer then produces a conventional printout or otherrecord of the final results, together with as much of the original dataas may be requested by the program. For example, the physician for whomthe analysis is being carried out may require a complete copy of all thedata on magnetic tape or the like for possible future reference. Also,photographs of the immunodiffusion plate can be taken, either directlyor using the monitor tube.

Ordinarily, one cycle of scanning does not permit an adequatequantitative determination of a protein unless it is present in highconcentration. After a suitable length of supplementary incubation,which may vary from a few minutes, say, to an hour or more, the abovedescribed scanning process is repeated, typically for the entire set oftest plates and the corresponding standards. The computer is typicallyinstructed to explore plate areas where zones were expected but notfound during the previous scan; and also areas corresponding to thepreviously found and recorded precipitation zones, preferably allowingfor specified expansion or movement of those zones which are set intothe program on the basis of previous experience. Thus, the computeroperations can maintain a continuity of treatment of the respectiveprotein zones between one scan and the next.

Occasionally two precipitation zones due to distinct proteins are soclose together that their normal growth ultimately produces overlap. Forzones produced by immunoelectrophoresis, such overlap normally occurs ator near the zone ends, leading to crossing of the zones as showntypically for zones a and b of FIG. 1D. When immunodiffusion is carriedout without initial fractionation, the overlap tends to be confined tothe zone center portions, as when zones d and d' of that figure growtogether. The computer is typically programmed to anticipate such a zoneoverlap, to recognize it when it has occurred, and then to make suitablemodifications in the procedure used for deriving the various parameters.

When the proteins under study are such that overlaps are expected, eachplate is typically scanned at an early stage of zone development beforezone overlaps have occurred (FIG. 1B). The zone axis and end points canthen be located without ambiguity. The computer is typically programmedas part of the regular processing of each scan to check for actualoverlaps, for example by comparing the (x,y) coordinates for eachmeasured point of a zone axis with those for the adjacent zones,coincidence within a specified threshold indicating an overlap. Allscans preferably also include a check for potential overlaps. Forexample, the computer extrapolates each zone axis beyond the observedend points and compares the extrapolated axis points of adjacent zones.Such axis extrapolation may comprise a simple linear extension in thedirection of the axis slope near each end. Alternatively, the observedzone axis may be fitted by a parabola or other curve, which can then beextrapolated accurately.

Zone overlap can also be detected by computing the rate of change ofslope of the zone axis, or the radius of curvature of that axis, at aseries of points along the zone. Any departure from the normally smoothvariation of those functions indicates that the zone may be made up oftwo or more overlapping zones.

When an actual overlap is found on one half of a zone, sufficientlyaccurate compensation can often be made by simply replacing the observedintensity values in the area of overlap with the values observed at thecorresponding points of the other half of the zone. Such correspondencebetween two points for zones due to immunoelectrophoresis is typicallydefined as equal x spacing on opposite sides of x_(g) of the point ofclosest approach to the antibody well; and for zones due to directimmunodiffusion is defined as equal y spacing on opposite sides of thezone axis. The axis within the area of overlap can usually be located byextrapolation.

More elaborate and accurate compensation procedures are also available,if desired. For example, the computer is instructed to adjust themeasurement at the selected symmetrically placed point of the unaffectedhalf of the zone to take account of any actual lack of symmetry of thezone. That unsymmetry can be determined, for example, by comparing thevalues that were obtained for the two points during a previous scanprior to the overlap, and applying the ratio of those values as acorrection factor.

A further illustrative compensation procedure takes account also of theintensity value actually measured at the region of overlap, which isdue, of course, partly to one zone and partly to the other. The computeris instructed to divide that observed value at each point of overlap inan appropriate ratio. A suitable approximate ratio may be obtained bycomparing measured values for the two zones at the respectivesymmetrically placed points already described, either with or withoutthe described adjustment of those values.

Whenever possible, it is preferred to employ two or more distinctcomputation procedures for compensating for areas of overlap, averagingthe results and determining the probable error. It is emphasized,however, that the region of overlap is ordinarily a small fraction ofthe entire area of a zone. Hence even quite appreciable errors inapproximating the true value within the overlap still may not affect thefinal result significantly.

Also, for each of the described types of overlap, it is usually possibleto make the determination of protein concentration in terms ofparameters that are defined by portions of the zone not affected by theoverlap. Thus, the measurements preferably employed are those near thezone center for processes similar to immunoelectrophoresis, and thosenear the zone ends for processes similar to direct immunodiffusion.

It will be recognized by those skilled in the art that many particularsof the described procedures can be replaced by their obvious equivalentswithout departing from the proper scope of the present invention. Forexample, values for those parameters that do not require a series ofmeasurements at successive incubation times can be obtained frommeasurements made after the incubation process has been allowed to reachsubstantial equilibrium. Such measurements have the advantage that thezone configurations are virtually static, and the exact time ofmeasurement is therefore not crucial. As a further example, at a desiredstage of the incubation the zones of precipitation can be stained inknown manner with an appropriate dye, and zone measurements can then bemade using selectively transmitted light. That method of measurement isusually most useful after equilibrium has been reached, since it is thennot necessary to assign a precise time of measurement.

In summary, methods have been described by which immunodiffusion,immunoelectrophoresis and analagous processes can be used to providetruly quantitative measurement of the concentrations of individualproteins in biological fluids. Actual use of the described methods hasdemonstrated that protein concentrations can be determined within fivepercent or better. The invention thus provides an effective andconvenient method for making a wide variety of experimental anddiagnostic determinations.

We claim:
 1. Apparatus for obtaining a quantitative measure of theconcentration of a protein in an antigen sample, comprisingmeans forsupporting and illuminating a plate carrying a precipitation zone due toimmunoelectrophoresis of said antigen sample with an antibody specificto said protein, photoresponsive means for extracting a plurality ofelectrical signals representing the light intensity at positions on saidplate which correspond to respective sets of position representingsignals, and electronic means for supplying to said photoresponsivemeans sets of position representing signals corresponding to positionsin selected respective spatial relationship to said zone, and forderiving from the resulting light intensity representing signals thecorresponding value of a parameter which varies in characteristic mannerwith said protein concentration.
 2. Apparatus according to claim 1wherein said electronic means includemeans for supplying automaticallyto said photoresponsive means a series of sets of position representingsignals which correspond to a two-dimensional array of positionsdistributed partly within and partly outside the zone, and memory meansfor storing the resulting light representing signals together withposition signals representing the position to which each lightrepresenting signal corresponds.
 3. Apparatus according to claim 1,includingalso means for producing a two-dimensional visual display ofsaid light representing signals, said electronic means includingmanuallycontrollable means for developing a set of position representing signalscorresponding to a selected position on said display, and means forproducing in digital form the light representing signal corresponding tothe so developed position representing signals.
 4. Apparatus forobtaining a quantitative measure of the concentration of an antigen inan antigen containing sample which has been subjected to immunodiffusionwith an antibody specific to said antigen, the antigen and antibodydiffusing through a supporting medium from respective sources mutuallyspaced along an axis and reacting to form at least one elongatedprecipitation zone of limited length; said apparatus comprisingmeans forautomatically scanning the supporting medium to develop electricalsignals representing light intensity at respective positions of atwo-dimensional array distributed partly within the zone and partlyoutside the zone, each light representing signal being associated withelectrical position representing signals identifying the correspondingposition in said array, and means responsive jointly to positionrepresenting signals and the corresponding light representing signalsfor deriving the value of a selected parameter of the zone which variesin characteristic manner with said antigen concentration.
 5. Apparatusaccording to claim 4 including means for causing said scanning means torepeat said scanning action intermittently, and means for associatingelectrical time signals with said light representing signals. 6.Apparatus for obtaining a quantitative measure of the concentration ofan antigen in an antigen containing sample by subjecting the sample toimmunodiffusion with an antibody source containing an antibody specificto said antigen, said reactants diffusing in a supporting medium fromrespective antigen and antibody wells to produce a precipitation zone;said apparatus comprisingphotoresponsive means for scanning the wellsand the supporting medium to develop electrical signals representinglight intensity at respective positions, each light representing signalbeing associated with electrical position representing signalsidentifying the corresponding position, dispensing means for dispensingantigen and antibody containing solutions selectively to the respectivewells in response to electrical command signals, and control meansresponsive jointly to the light representing signals and the positionrepresenting signals and having one mode of operation for producingoperative position of the dispensing means relative to each individualwell and for supplying a command signal to the dispensing means tosupply that well with a selected reagent, said control means havinganother mode of operation for deriving electronically from the lightrepresenting signals and the position representing signals thecorresponding value of a selected zone parameter which varies incharacteristic manner with said antigen concentration.
 7. Apparatusaccording to claim 6, said control means, when in said one mode,including means for deriving signals representing the position of eachwell, and being responsive to such well position signals for producingoperative position of the dispensing means.
 8. Apparatus according toclaim 6 includingmeans associated with said control means, when in saidone mode, for comparing the relative positions of said wells withpredetermined standard relative well positions to detect deviationstherefrom, and means associated with said control means, when in saidother mode, for compensating for such deviations during said parameterderivation.